Splice Site Cleavage Research Articles

Exploitation of a Thermosensitive Splicing Event To Study Pre-mRNA Splicing In Vivo

The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33 degrees C or lower than at 37 to 41 degrees C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41 degrees C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. We exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39 degrees C. However, after a short (about 30-min) lag following a shift to 33 degrees C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA. Increased splicing at this site in 54-5A4 viral RNA is probably driven by the unavailability of the usual 3' splice site for exon ligation. The thermosensitivity of this alternate splice event suggests that the sequences governing the thermodependence of MuSVts110 RNA splicing do not involve any particular 3' splice site or branch point sequence, but rather lie near the 5' end of the intron.

Role of the 3' splice site consensus sequence in mammalian pre-mRNA splicing.

Pre-mRNA splicing has been shown to occur by a two-step pathway. In the first stage, the pre-mRNA is cleaved at the 5' splice site, generating the first exon RNA species and an RNA species composed of the intron and second exon (IVS1-exon 2 RNA species). In the second stage, cleavage at the 3' splice site and ligation of the exons occurs, resulting in the excision of the intact intron. The excised intron and IVS1-exon 2 RNA species are in the form of a lariat in which the 5' end of the intron is joined to an adenosine residue near the 3' end of the intron by a 2'-5' phosphodiester bond. Here we show that although cleavage at the 3' splice site does not occur until the second stage of the splicing reaction, at least a portion of the 3' splice site consensus sequence is necessary for 5' splice site cleavage and lariat formation. Thus, in higher eukaryotes at least three sequence elements participate in the initiation of the splicing reaction: the 5' splice site, 3' splice site consensus sequence and the RNA branchpoint.